Peroxidase-producing actinobacteria from Algerian environments and insights from the genome sequence of peroxidase-producing Streptomyces sp. S19 / Actinobacteria productora de peroxidasa de ambientes argelinos y perspectivas de la secuencia genómica de Streptomyces sp. S19

Unique environments often serve as a source of novel microorganisms with novel chemistries. In this study, telluric samples collected from different regions of Algeria were processed for the isolation of novel peroxidase-producing actinobacterial strains. An agar-based screening identified 45 isolates with the ability to produce peroxidase. The 16S rRNA gene sequencing showed that most of the strains belong to the genus Streptomyces. Optimization of cultivation conditions was performed for the top five peroxidase-producing strains. Apart from strain 36 (optimal growth temperature of 30 °C) and strain 45 (optimal medium pH of 6.0), the strains exhibited optimal peroxidase production when cultivated for 5 days at 37 °C and in a medium at pH 7.0. Extracellular peroxidase production was induced by ferulic acid in three of the five strains, while the presence of canola lignocellulosic waste (CLW) induced peroxidase production in all strains. Strain 19 (S19) was selected for further optimization and the extracellular peroxidase purified using acid and acetone precipitation, followed by size exclusion chromatography. The purified fraction showed a single band on the polyacrylamide gel with an estimated molecular weight of 21.45 kDa. Genome analysis confirmed the assignment of S19 to the genus Streptomyces, the presence of genes encoding for peroxidases, and the presence of genes encoding for carbohydrate-active enzymes. The presence of biosynthetic gene clusters potentially encoding for biosurfactants further highlighted the great biotechnological potential of the strain.(AU)

Peroxidase-producing actinobacteria from Algerian environments and insights from the genome sequence of peroxidase-producing Streptomyces sp. S19.

Unique environments often serve as a source of novel microorganisms with novel chemistries. In this study, telluric samples collected from different regions of Algeria were processed for the isolation of novel peroxidase-producing actinobacterial strains. An agar-based screening identified 45 isolates with the ability to produce peroxidase. The 16S rRNA gene sequencing showed that most of the strains belong to the genus Streptomyces. Optimization of cultivation conditions was performed for the top five peroxidase-producing strains. Apart from strain 36 (optimal growth temperature of 30 °C) and strain 45 (optimal medium pH of 6.0), the strains exhibited optimal peroxidase production when cultivated for 5 days at 37 °C and in a medium at pH 7.0. Extracellular peroxidase production was induced by ferulic acid in three of the five strains, while the presence of canola lignocellulosic waste (CLW) induced peroxidase production in all strains. Strain 19 (S19) was selected for further optimization and the extracellular peroxidase purified using acid and acetone precipitation, followed by size exclusion chromatography. The purified fraction showed a single band on the polyacrylamide gel with an estimated molecular weight of 21.45 kDa. Genome analysis confirmed the assignment of S19 to the genus Streptomyces, the presence of genes encoding for peroxidases, and the presence of genes encoding for carbohydrate-active enzymes. The presence of biosynthetic gene clusters potentially encoding for biosurfactants further highlighted the great biotechnological potential of the strain.

Optimization of ß-1,4-Endoxylanase Production by an Aspergillus niger Strain Growing on Wheat Straw and Application in Xylooligosaccharides Production.

Plant biomass constitutes the main source of renewable carbon on the planet. Its valorization has traditionally been focused on the use of cellulose, although hemicellulose is the second most abundant group of polysaccharides on Earth. The main enzymes involved in plant biomass degradation are glycosyl hydrolases, and filamentous fungi are good producers of these enzymes. In this study, a new strain of Aspergillus niger was used for hemicellulase production under solid-state fermentation using wheat straw as single-carbon source. Physicochemical parameters for the production of an endoxylanase were optimized by using a One-Factor-at-a-Time (OFAT) approach and response surface methodology (RSM). Maximum xylanase yield after RSM optimization was increased 3-fold, and 1.41- fold purification was achieved after ultrafiltration and ion-exchange chromatography, with about 6.2% yield. The highest activity of the purified xylanase was observed at 50 °C and pH 6. The enzyme displayed high thermal and pH stability, with more than 90% residual activity between pH 3.0-9.0 and between 30-40 °C, after 24 h of incubation, with half-lives of 30 min at 50 and 60 °C. The enzyme was mostly active against wheat arabinoxylan, and its kinetic parameters were analyzed (Km = 26.06 mg·mL-1 and Vmax = 5.647 U·mg-1). Wheat straw xylan hydrolysis with the purified ß-1,4 endoxylanase showed that it was able to release xylooligosaccharides, making it suitable for different applications in food technology.

α-Amylase production by Tepidimonas fonticaldi strain HB23: statistical optimization and compatibility study for use in detergent formulations.

In a previous study, a thermostable α-amylase-producing bacterium (designated HB23) was isolated from an Algerian hydrothermal spring. In the present study, the native strain was subjected to a statistical optimization aimed at enhancing the α-amylase production. To achieve this, thirteen factors have been studied, among which are cultural and nutritional parameters. Wheat bran, a by-product of the grain milling industry, was the factor that positively influenced α-amylase production. A modified L27 Taguchi design was used to screen these factors. Furthermore, a Box-Behnken matrix, supplemented by the use of response surface methodology (RSM), allowed for the identification of optimum levels of the following factors: a 1% inoculum size, 15 g/L soluble starch, 5 g/L wheat bran, and 1 g/L tryptone. Optimized conditions resulted in an amylolytic activity of 320 U/mL, which is a tenfold increase when compared with unoptimized production level. Phenotypical and molecular identification of strain HB23 revealed its close relationship to various Tepidimonas strains, specifically to Tepidimonas fonticaldi. The crude enzyme preparation turned out to be compatible with various laundry detergents and led to a substantial improvement in their washing performance. A comparison of the performance of the crude enzyme preparation with that of the commercial α-amylase (Termamyl® 300 L) highlights the potential of the HB23 enzyme as a bio-additive in detergent formulations.

Differential antioxidant activity of glucuronoxylooligosaccharides (UXOS) and arabinoxylooligosaccharides (AXOS) produced by two novel xylanases.

XOS are particularly interesting bioactive molecules. Bacillus safensis CBLMA18, a xylanolytic bacterium has been isolated and two of its xylanases have been identified and fully characterized. Xyn11A is an extracellular 22.5-kDa GH11 xylanase while a second xylanase, Xyn10B, corresponds to an intracellular 48-kDa GH10 enzyme. Both unimodular xylanases showed activity only on xylan substrates with important differences in their catalytic pattern. Xyn11A displays higher activity on glucuronoxylans, with an optimum at pH 8 and 50 °C, and a Vmax of 5281 U/mg on beechwood xylan, meanwhile Xyn10B prefers arabinoxylans, with an optimum of pH 7 and 60 °C, and a Vmax of 50.29 U/mg on rye arabinoxylan. The antioxidant activity of xylanase-generated XOS obtained from glucuronoxylans (UXOS) and arabinoxylans (AXOS) was tested with the ABTS (2, 2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) method. UXOS showed higher antioxidant activity than AXOS (>80% of antioxidant capacity). Thin layer chromatography and MALDI-TOF MS analysis showed that UXOS comprise neutral and acidic XOS with methylglucuronic acid (MeGlcA) ramifications, while AXOS contain only neutral molecules with arabinose decorations. The MeGlcA ramifications seem to have an important role in the antioxidant capacity of oligosaccharides. Besides, the increase of UXOS size correlates with an increase in their activity.

Purification, biochemical, and molecular characterization of a novel extracellular thermostable and alkaline α-amylase from Tepidimonas fonticaldi strain HB23.

The present study investigated the purification, biochemical, and molecular characterization of a novel thermostable α-amylase (TfAmy48) from Tepidimonas fonticaldi strain HB23. MALDI-TOF/MS analysis indicated that the purified enzyme is a monomer with a molecular mass of 48,138.10 Da. The results from amino-acid sequence analysis revealed high homology between the 25 NH2-terminal residues of TfAmy48 and those of Gammaproteobacteria α-amylases. The optimum pH and temperature values for α-amylase activity were pH 8 and 80 °C, respectively. Thin-layer chromatography (TLC) analysis showed that the final hydrolyzed products of the enzyme from soluble potato starch were maltopentaose, maltose, and maltotriose, which indicate that TfAmy48 possessed an endo-acting pattern. Compared to Termamyl®300 L, TfAmy48 showed extreme stability and tolerance towards organic solvents and excellent compatibility with some commercial laundry detergents. These proprieties make TfAmy48 enzyme a potential candidate as a cleaning bioadditive in detergent composition. The Tfamy48 gene encoding TfAmy48 was cloned, sequenced, and heterologously-expressed in the extracellular fraction of Escherichia coli strain BL21(DE3)pLysS. The biochemical properties of the extracellular purified recombinant enzyme (rTfAmy48) were similar to those of native one. The highest sequence identity value (97%) was obtained with PsAmy1 α-amylase from Pseudomonas sp. strain KFCC10818, with only 16 amino-acid (aa) residues of difference.

Cellulase-Hemicellulase Activities and Bacterial Community Composition of Different Soils from Algerian Ecosystems.

Soil microorganisms are important mediators of carbon cycling in nature. Although cellulose- and hemicellulose-degrading bacteria have been isolated from Algerian ecosystems, the information on the composition of soil bacterial communities and thus the potential of their members to decompose plant residues is still limited. The objective of the present study was to describe and compare the bacterial community composition in Algerian soils (crop, forest, garden, and desert) and the activity of cellulose- and hemicellulose-degrading enzymes. Bacterial communities were characterized by high-throughput 16S amplicon sequencing followed by the in silico prediction of their functional potential. The highest lignocellulolytic activity was recorded in forest and garden soils whereas activities in the agricultural and desert soils were typically low. The bacterial phyla Proteobacteria (in particular classes α-proteobacteria, δ-proteobacteria, and γ-proteobacteria), Firmicutes, and Actinobacteria dominated in all soils. Forest and garden soils exhibited higher diversity than agricultural and desert soils. Endocellulase activity was elevated in forest and garden soils. In silico analysis predicted higher share of genes assigned to general metabolism in forest and garden soils compared with agricultural and arid soils, particularly in carbohydrate metabolism. The highest potential of lignocellulose decomposition was predicted for forest soils, which is in agreement with the highest activity of corresponding enzymes.

Isolation and characterization from solar salterns of North Algeria of a haloarchaeon producing a new halocin.

Halophilic archaea, thriving in hypersaline environments, synthesize antimicrobial substances with an unknown role, called halocins. It has been suggested that halocin production gives transient competitive advantages to the producer strains and represents one of the environmental factors influencing the microbial community composition. Herein, we report on the antibacterial activity of a new haloarchaeon selected from solar salterns of the northern coast of Algeria. A total of 81 halophilic strains, isolated from the microbial consortia, were screened for the production of antimicrobial compounds by interspecies competition test and against a collection of commercial haloarchaea. On the basis of the partial 16S rRNA sequencing, the most efficient halocin producer was recognized as belonging to Haloferax (Hfx) sp., while the best indicator microorganism, showing high sensitivity toward halocin, was related to Haloarcula genus. The main morphological, physiological and biochemical properties of Hfx were investigated and a partial purification of the produced halocin was allowed to identify it as a surface membrane protein with a molecular mass between 30 and 40 kDa. Therefore, in this study, we isolated a new strain belonging to Haloferax genus and producing a promising antimicrobial compound useful for applications in health and food industries.

Biochemical properties of a new thermo- and solvent-stable xylanase recovered using three phase partitioning from the extract of Bacillus oceanisediminis strain SJ3.

The present study investigates the production and partial biochemical characterization of an extracellular thermostable xylanase from the Bacillus oceanisediminis strain SJ3 newly recovered from Algerian soil using three phase partitioning (TPP). The maximum xylanase activity recorded after 2 days of incubation at 37 °C was 20.24 U/ml in the presence of oat spelt xylan. The results indicated that the enzyme recovered in the middle phase of TPP system using the optimum parameters were determined as 50% ammonium sulfate saturation with 1.0:1.5 ratio of crude extract: t-butanol at pH and temperature of 8.0 and 10 °C, respectively. The xylanase was recovered with 3.48 purification fold and 107% activity recovery. The enzyme was optimally active at pH 7.0 and was stable over a broad pH range of 5.0-10. The optimum temperature for xylanase activity was 55 °C and the half-life time at this temperature was of 6 h. At this time point the enzyme retained 50% of its activity after incubation for 2 h at 95 °C. The crude enzyme resist to sodium dodecyl sulfate and ß-mercaptoethanol, while all the tested ions do not affect the activity of the enzyme. The recovered enzyme is, at least, stable in tested organic solvents except in propanol where a reduction of 46.5% was observed. Further, the stability of the xylanase was higher in hydrophobic solvents where a maximum stability was observed with cyclohexane. These properties make this enzyme to be highly thermostable and may be suggested as a potential candidate for application in some industrial processes. To the best of our knowledge, this is the first report of xylanase activity and recoverey using three phase partitioning from B. oceanisediminis.

Efficient screening of potential cellulases and hemicellulases produced by Bosea sp. FBZP-16 using the combination of enzyme assays and genome analysis.

Identification of bacteria that produce carbohydrolytic enzymes is extremely important given the increased demand for these enzymes in many industries. Twenty lignocellulose-degrading bacterial isolates from Algerian compost and different soils were screened for their potential to produce different enzymes involved in biomass deconstruction. Based on 16S rRNA gene sequencing, the isolates belonged to Proteobacteria and Actinobacteria. Differences among species were reflected both as the presence/absence of enzymes or at the level of enzyme activity. Among the most active species, Bosea sp. FBZP-16 demonstrated cellulolytic activity on both amorphous cellulose (CMC) and complex lignocellulose (wheat straw) and was selected for whole-genomic sequencing. The genome sequencing revealed the presence of a complex enzymatic machinery required for organic matter decomposition. Analysis of the enzyme-encoding genes indicated that multiple genes for endoglucanase, xylanase, ß-glucosidase and ß-mannosidase are present in the genome with enzyme activities displayed by the bacterium, while other enzymes, such as certain cellobiohydrolases, were not detected at the genomic level. This indicates that a combination of functional screening of bacterial cultures with the use of genome-derived information is important for the prediction of potential enzyme production. These results provide insight into their possible exploitation for the production of fuels and chemicals derived from plant biomass.

Staphylococcal cassette chromosome mec typing and mecA sequencing in methicillin-resistant staphylococci from Algeria: a highly diversified element with new mutations in mecA.

Genetic mechanisms of methicillin resistance are still relevant in staphylococci. The aims of this study are to assess the possible exchanges of staphylococcal cassette chromosome mec (SCCmec) among isolates of methicillin-resistant staphylococci (MRS) and to check for known or new mutations in mecA DNA. A total of 35 MRS non-repetitive isolates were recovered, including 20 Staphylococcushaemolyticus, 7 Staphylococcusaureus, 4 Staphylococcussciuri, 2 Staphylococcussaprophyticus and 1 isolate each of Staphylococcusxylosus and Staphylococcuslentus. Only 16 of the 35 strains were assigned to known SCCmec types: 7 SCCmec VII, 6 SCCmec IV and 3 SCCmec III, with possible horizontal transfer of the SCCmec VII from methicillin-resistant S. haemolyticus to methicillin-susceptible S. aureus. mecA gene sequencing in ten selected isolates allowed description of nine punctual mutations, seven of which were reported for the first time. The most frequent mutation was G246E, identified in isolates of methicillin-resistant S. aureus, S. sciuri, S. saprophyticus and S. lentus. These results emphasized the high degree of genetic diversity of SCCmec element in MRS and describe new missense mutations in mecA, which might be important in understanding the evolution of methicillin and new ß-lactam resistance.

Descriptive epidemiology of nasal carriage of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus among patients admitted to two healthcare facilities in Algeria.

AIM: To evaluate nasal carriage rate and variables associated with Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) in patients admitted in two healthcare facilities. RESULTS: S. aureus was isolated from 159 (26%) of the enrolled patients. Methicillin-susceptible S. aureus was isolated from 150 (24.5%) patients, and MRSA was isolated from 9 (1.5%). Cancer and previous hospitalization were associated with a significantly higher frequency of nasal S. aureus carriage among the patients admitted to the general hospital and the nephrology department, respectively. MRSA isolates were heterogeneous with respect to their staphylococcal cassette chromosome mec element (SCCmec) type, sequence type (ST), and toxin genes (pvl and tst1) content. Four isolates were attributed with the ST80-MRSA-IV clone, which is known to be predominant in Algeria. CONCLUSIONS: This is the first assessment of S. aureus and MRSA nasal carriage and associated variables in Algeria. Our findings provide also a picture of the MRSA strains circulating in the community in this geographic area. They can be useful as a guide for implementing screening and control procedures against S. aureus/MRSA in the Algerian healthcare facilities.

Partial characterization of xylanase produced by Caldicoprobacter algeriensis, a new thermophilic anaerobic bacterium isolated from an Algerian hot spring.

To date, xylanases have expanded their use in many processing industries, such as pulp, paper, food, and textile. This study aimed the production and partial characterization of a thermostable xylanase from a novel thermophilic anaerobic bacterium Caldicoprobacter algeriensis strain TH7C1(T) isolated from a northeast hot spring in Algeria. The obtained results showed that C. algeriensis xylanase seems not to be correlated with the biomass growth profile whereas the maximum enzyme production (140.0 U/ml) was recorded in stationary phase (18 h). The temperature and pH for optimal activities were 70 °C and 11.0, respectively. The enzyme was found to be stable at 50, 60, 70, and 80 °C, with a half-life of 10, 9, 8, and 4 h, respectively. Influence of metal ions on enzyme activity revealed that Ca(+2) enhances greatly the relative activity to 151.3 %; whereas Hg(2+) inhibited significantly the enzyme. At the best of our knowledge, this is the first report on the production of xylanase by the thermophilic bacterium C. algeriensis. This thermo- and alkaline-tolerant xylanase could be used in pulp bleaching process.

Purification and characterization of the xylanase produced by Jonesia denitrificans BN-13.

Jonesia denitrificans BN-13 produces six xylanases: Xyl1, Xyl2, Xyl3, Xyl4, Xyl5, and Xyl6; the Xyl4 was purified and characterized after two consecutive purification steps using ultrafiltration and anion exchange chromatography. The xylanase-specific activity was found to be 77 unit (U)/mg. The molecular weight of the Xyl4 estimated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a monomeric isoenzyme of about 42 kDa. It showed an optimum pH value of 7.0 and a temperature of 50 °C. It was stable at 50 °C for 9.34 h. The enzyme showed to be activated by Mn(+2), ß-mercaptoethanol, and dithiothreitol (DTT) with a high affinity towards birchwood xylan (with a K(m) of 1 mg ml(-1)) and hydrolysis of oat-spelt xylan with a K(m) of 1.85 mg ml(-1). The ability of binding to cellulose and/or xylan was also investigated.

Panton-Valentine leukocidin positive sequence type 80 methicillin-resistant Staphylococcus aureus carrying a staphylococcal cassette chromosome mec type IVc is dominant in neonates and children in an Algiers hospital.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major antimicrobial drug-resistant pathogen causing serious infections. It was first detected in healthcare settings, but in recent years it has also become disseminated in the community. Children and young adults are most susceptible to infection by community-acquired (CA) MRSA strains. In this study 25 MRSA isolates implicated in infections of neonates and children admitted to an Algiers hospital during an 18 month period were characterized by molecular methods including staphylococcal cassette chromosome (SCC) mec typing, PCR amplification of pvl genes, pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Fifteen out of 25 isolates were from hospital-acquired infections. Twenty-four isolates carried SCCmec type IVc and belonged to the sequence type (ST) 80, one isolate carried SCCmec type II and was ST 39. Twenty-two out of 24 ST80-MRSA-IVc isolates carried pvl genes. Our results suggest that the Panton-Valentine leukocidin positive ST80- MRSA-IVc is the dominant MRSA clone causing disease in neonates and children in Algiers.

Retama species growing in different ecological-climatic areas of northeastern Algeria have a narrow range of rhizobia that form a novel phylogenetic clade within the Bradyrhizobium genus.

Sixty-seven isolates were isolated from nodules collected on roots of Mediterranean shrubby legumes Retama raetam and Retama sphaerocarpa growing in seven ecological-climatic areas of northeastern Algeria. Genetic diversity of the Retama isolates was analyzed based on genotyping by restriction fragment length polymorphism of PCR-amplified fragments of the 16S rRNA gene, the intergenic spacer (IGS) region between the 16S and 23S rRNA genes (IGS), and the symbiotic genes nifH and nodC. Eleven haplotypes assigned to the Bradyrhizobium genus were identified. Significant biogeographical differentiation of the rhizobial populations was found, but one haplotype was predominant and conserved across the sites. All isolates were able to cross-nodulate the two Retama species. Accordingly, no significant genetic differentiation of the rhizobial populations was found in relation to the host species of origin. Sequence analysis of the 16S rRNA gene grouped the isolates with Bradyrhizobium elkanii, but sequence analyses of IGS, the housekeeping genes (dnaK, glnII, recA), nifH, and nodC yielded convergent results showing that the Retama nodule isolates from the northeast of Algeria formed a single evolutionary lineage, which was well differentiated from the currently named species or well-delineated unnamed genospecies of bradyrhizobia. Therefore, this study showed that the Retama species native to northeastern Algeria were associated with a specific clade of bradyrhizobia. The Retama isolates formed three sub-groups based on IGS and housekeeping gene phylogenies, which might form three sister species within a novel bradyrhizobial clade.

First report of qnrB-producing Enterobacter cloacae and qnrA-producing Acinetobacter baumannii recovered from Algerian hospitals.

Two isolates of Enterobacter cloacae and 1 isolate of Acinetobacter baumannii showing a multidrug resistance phenotype including resistance to beta-lactams and fluoroquinolones were included in this study. By polymerase chain reaction amplification and sequencing, the 2 isolates of E. cloacae were found to produce QnrB and the A. baumannii isolate was found to produce QnrA. In addition, the 2 E. cloacae isolates were found to produce CTX-M-15.

Characterization of CTX-M-15-producing Klebsiella pneumoniae and Escherichia coli strains isolated from hospital environments in Algeria.

For detecting extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in the hospital environment, sedimentation plates were placed in the rooms of two hospitals. Three environmental isolates, two Klebsiella pneumoniae, and one Escherichia coli resistant to extended-spectrum cephalosporins with a phenotype indicating CTX-M enzymes production (the minimum inhibitory concentration [MIC] of cefotaxime was higher than the MIC of ceftazidime) were recovered. By PCR and sequencing, the three isolates were found to produce CTX-M-15. The bla(CTX-M-15) genes in the three isolates were transferred by conjugation. One K. pneumoniae environmental isolate showed an identical and unique RAPD profile with two other K. pneumoniae clinical isolates recovered from urinary tract infection from patients hospitalized in two different wards of another hospital.

Biochemical and phylogenetic characterization of a novel terrestrial hyperthermophilic archaeon pertaining to the genus Pyrococcus from an Algerian hydrothermal hot spring.

A hyperthermophilic anaerobic archeon, strain HT3, was isolated from hydrothermal hot spring in Northeast Algeria. The strain is a regular coccus, highly motile, obligatory anaerobic, heterotrophic. It utilizes proteinaceous complex media (peptone, tryptone or yeast extract). Sulfur is reduced to Hydrogen sulfide and enhances growth. It shares with other Pyrococcus species the heterotrophic mode of nutrition, the hyperthermophily, the ability to utilize amino acids as sole carbon and nitrogen sources and the ether lipid composition. The optimal growth occurs at 80-85 degrees C, pH 7.5 and 1.5% NaCl. The G + C content was 43 mol%. Considering its morphology, physiological properties, nutritional features and phylogenetic analyses based on 16S rRNA gene sequencing, this strain is described as a new terrestrial isolate pertaining to the genus Pyrococcus.